RESUMEN
Classical swine fever (CSF) is caused by the classical swine fever virus (CSFV), which poses a threat to swine production. The activation of host innate immunity through linker proteins such as tumor necrosis factor receptor (TNF-R)-associated factor (TRAF) is crucial for the induction of the NF-κB pathway. Recent research has revealed the involvement of mitochondrial antiviral-signaling protein (MAVS) in the interaction with TRAF2, 3, 5, and 6 to activate both the NF-κB and IRF3 pathways. This study revealed that CSFV infection led to the upregulation of TRAF1 mRNA and protein levels; moreover, TRAF1 overexpression inhibited CSFV replication, while TRAF1 knockdown promoted replication, highlighting its importance in the host response to CSFV infection. Additionally, the expression of RIG-I, MAVS, TRAF1, IRF1, and ISG15 were detected in PK-15 cells infected with CSFV, revealing that TRAF1 plays a role in regulating IRF1 and ISG15 within the RIG-I pathway. Furthermore, Co-IP, GST pull-down, and IFA analyses demonstrated that TRAF1 interacted with MAVS and co-localized in the cytoplasm during CSFV infection. Ultimately, TRAF1 acted as a novel member of the TRAF family, bound to MAVS as a linker molecule, and functioned as a mediator downstream of MAVS in the RIG-I/MAVS pathway against CSFV replication.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Virus de la Fiebre Porcina Clásica , Factor 1 Regulador del Interferón , Factor 1 Asociado a Receptor de TNF , Regulación hacia Arriba , Animales , Virus de la Fiebre Porcina Clásica/fisiología , Factor 1 Asociado a Receptor de TNF/metabolismo , Factor 1 Asociado a Receptor de TNF/genética , Porcinos , Regulación hacia Arriba/genética , Factor 1 Regulador del Interferón/metabolismo , Factor 1 Regulador del Interferón/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Transducción de Señal , Peste Porcina Clásica/virología , Peste Porcina Clásica/metabolismo , Peste Porcina Clásica/genética , Replicación Viral , Línea Celular , Citocinas/metabolismo , Unión ProteicaRESUMEN
Mitochondria are energy producers in cells, which can affect viral replication by regulating the host innate immune signaling pathways, and the changes in their biological functions are inextricably linked the viral life cycle. In this study, we screened a library of 382 mitochondria-targeted compounds and identified the antiviral inhibitors of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme in the de novo synthesis pathway of pyrimidine ribonucleotides, against classical swine fever virus (CSFV). Our data showed that the inhibitors interfered with viral RNA synthesis in a dose-dependent manner, with half-maximal effective concentrations (EC50) ranging from 0.975 to 26.635 nM. Remarkably, DHODH inhibitors obstructed CSFV replication by enhancing the innate immune response including the TBK1-IRF3-STAT1 and NF-κB signaling pathways. Furthermore, the data from a series of compound addition and supplementation trials indicated that DHODH inhibitors also inhibited CSFV replication by blocking the de novo pyrimidine synthesis. Remarkably, DHODH knockdown demonstrated that it was essential for CSFV replication. Mechanistically, confocal microscopy and immunoprecipitation assays showed that the non-structural protein 4A (NS4A) recruited and interacted with DHODH in the perinuclear. Notably, NS4A enhanced the DHODH activity and promoted the generation of UMP for efficient viral replication. Structurally, the amino acids 65-229 of DHODH and the amino acids 25-40 of NS4A were pivotal for this interaction. Taken together, our findings highlight the critical role of DHODH in the CSFV life cycle and offer a potential antiviral target for the development of novel therapeutics against CSF. IMPORTANCE: Classical swine fever remains one of the most economically important viral diseases of domestic pigs and wild boar worldwide. dihydroorotate dehydrogenase (DHODH) inhibitors have been shown to suppress the replication of several viruses in vitro and in vivo, but the effects on Pestivirus remain unknown. In this study, three specific DHODH inhibitors, including DHODH-IN-16, BAY-2402234, and Brequinar were found to strongly suppress classical swine fever virus (CSFV) replication. These inhibitors target the host DHODH, depleting the pyrimidine nucleotide pool to exert their antiviral effects. Intriguingly, we observed that the non-structural protein 4A of CSFV induced DHODH to accumulate around the nucleus in conjunction with mitochondria. Moreover, NS4A exhibited a strong interaction with DHODH, enhancing its activity to promote efficient CSFV replication. In conclusion, our findings enhance the understanding of the pyrimidine synthesis in CSFV infection and expand the novel functions of CSFV NS4A in viral replication, providing a reference for further exploration of antiviral targets against CSFV.
Asunto(s)
Antivirales , Virus de la Fiebre Porcina Clásica , Dihidroorotato Deshidrogenasa , Proteínas no Estructurales Virales , Replicación Viral , Animales , Antivirales/farmacología , Antivirales/uso terapéutico , Línea Celular , Peste Porcina Clásica/tratamiento farmacológico , Peste Porcina Clásica/inmunología , Peste Porcina Clásica/metabolismo , Peste Porcina Clásica/virología , Virus de la Fiebre Porcina Clásica/efectos de los fármacos , Virus de la Fiebre Porcina Clásica/crecimiento & desarrollo , Virus de la Fiebre Porcina Clásica/inmunología , Virus de la Fiebre Porcina Clásica/metabolismo , Dihidroorotato Deshidrogenasa/metabolismo , Relación Dosis-Respuesta a Droga , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/inmunología , Inmunoprecipitación , Microscopía Confocal , Mitocondrias/enzimología , Mitocondrias/metabolismo , ARN Viral/biosíntesis , Transducción de Señal/efectos de los fármacos , Porcinos/virología , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Tryptophan metabolism plays a crucial role in facilitating various cellular processes essential for maintaining normal cellular function. Indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the conversion of tryptophan (Trp) into kynurenine (Kyn), thereby initiating the degradation of Trp. The resulting Kyn metabolites have been implicated in the modulation of immune responses. Currently, the role of IDO1-mediated tryptophan metabolism in the process of viral infection remains relatively unknown. In this study, we discovered that classical swine fever virus (CSFV) infection of PK-15 cells can induce the expression of IDO1, thereby promoting tryptophan metabolism. IDO1 can negatively regulate the NF-κB signaling by mediating tryptophan metabolism, thereby facilitating CSFV replication. We found that silencing the IDO1 gene enhances the expression of IFN-α, IFN-ß, and IL-6 by activating the NF-κB signaling pathway. Furthermore, our observations indicate that both silencing the IDO1 gene and administering exogenous tryptophan can inhibit CSFV replication by counteracting the cellular autophagy induced by Rapamycin. This study reveals a novel mechanism of IDO1-mediated tryptophan metabolism in CSFV infection, providing new insights and a theoretical basis for the treatment and control of CSFV.IMPORTANCEIt is well known that due to the widespread use of vaccines, the prevalence of classical swine fever (CSF) is shifting towards atypical and invisible infections. CSF can disrupt host metabolism, leading to persistent immune suppression in the host and causing significant harm when co-infected with other diseases. Changes in the host's metabolic profiles, such as increased catabolic metabolism of amino acids and the production of immunoregulatory metabolites and their derivatives, can also influence virus replication. Mammals utilize various pathways to modulate immune responses through amino acid utilization, including increased catabolic metabolism of amino acids and the production of immunoregulatory metabolites and their derivatives, thereby limiting viral replication. Therefore, this study proposes that targeting the modulation of tryptophan metabolism may represent an effective approach to control the progression of CSF.
Asunto(s)
Virus de la Fiebre Porcina Clásica , Indolamina-Pirrol 2,3,-Dioxigenasa , FN-kappa B , Transducción de Señal , Triptófano , Replicación Viral , Triptófano/metabolismo , Animales , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , FN-kappa B/metabolismo , Porcinos , Virus de la Fiebre Porcina Clásica/fisiología , Línea Celular , Quinurenina/metabolismo , Peste Porcina Clásica/virología , Peste Porcina Clásica/metabolismo , AutofagiaRESUMEN
Viruses exploit the host cell's energy metabolism system to support their replication. Mitochondria, known as the powerhouse of the cell, play a critical role in regulating cell survival and virus replication. Our prior research indicated that the classical swine fever virus (CSFV) alters mitochondrial dynamics and triggers glycolytic metabolic reprogramming. However, the role and mechanism of PKM2, a key regulatory enzyme of glycolytic metabolism, in CSFV replication remain unclear. In this study, we discovered that CSFV enhances PKM2 expression and utilizes PKM2 to inhibit pyruvate production. Using an affinity purification coupled mass spectrometry system, we successfully identified PKM as a novel interaction partner of the CSFV non-structural protein NS4A. Furthermore, we validated the interaction between PKM2 and both CSFV NS4A and NS5A through co-immunoprecipitation and confocal analysis. PKM2 was found to promote the expression of both NS4A and NS5A. Moreover, we observed that PKM2 induces mitophagy by activating the AMPK-mTOR signaling pathway, thereby facilitating CSFV proliferation. In summary, our data reveal a novel mechanism whereby PKM2, a metabolic enzyme, promotes CSFV proliferation by inducing mitophagy. These findings offer a new avenue for developing antiviral strategies. IMPORTANCE: Viruses rely on the host cell's material-energy metabolic system for replication, inducing host metabolic disorders and subsequent immunosuppression-a major contributor to persistent viral infections. Classical swine fever virus (CSFV) is no exception. Classical swine fever is a severe acute infectious disease caused by CSFV, resulting in significant economic losses to the global pig industry. While the role of the metabolic enzyme PKM2 (pyruvate dehydrogenase) in the glycolytic pathway of tumor cells has been extensively studied, its involvement in viral infection remains relatively unknown. Our data unveil a new mechanism by which the metabolic enzyme PKM2 mediates CSFV infection, offering novel avenues for the development of antiviral strategies.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Virus de la Fiebre Porcina Clásica , Mitofagia , Piruvato Quinasa , Serina-Treonina Quinasas TOR , Proteínas no Estructurales Virales , Replicación Viral , Animales , Proteínas Quinasas Activadas por AMP/metabolismo , Antivirales , Peste Porcina Clásica/metabolismo , Peste Porcina Clásica/virología , Virus de la Fiebre Porcina Clásica/crecimiento & desarrollo , Virus de la Fiebre Porcina Clásica/fisiología , Diseño de Fármacos , Glucólisis , Piruvato Quinasa/química , Piruvato Quinasa/metabolismo , Piruvatos/metabolismo , Transducción de Señal , Porcinos/metabolismo , Porcinos/virología , Serina-Treonina Quinasas TOR/metabolismo , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismoRESUMEN
IMPORTANCE: Of the flaviviruses, only CSFV and bovine viral diarrhea virus express Npro as the non-structural protein which is not essential for viral replication but functions to dampen host innate immunity. We have deciphered a novel mechanism with which CSFV uses to evade the host antiviral immunity by the N-terminal domain of its Npro to facilitate proteasomal degradation of Sp1 with subsequent reduction of HDAC1 and ISG15 expression. This is distinct from earlier findings involving Npro-mediated IRF3 degradation via the C-terminal domain. This study provides insights for further studies on how HDAC1 plays its role in antiviral immunity, and if and how other viral proteins, such as the core protein of CSFV, the nucleocapsid protein of porcine epidemic diarrhea virus, or even other coronaviruses, exert antiviral immune responses via the Sp1-HDAC1 axis. Such research may lead to a deeper understanding of viral immune evasion strategies as part of their pathogenetic mechanisms.
Asunto(s)
Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica , Endopeptidasas , Histona Desacetilasa 1 , Inmunidad Innata , Complejo de la Endopetidasa Proteasomal , Factor de Transcripción Sp1 , Proteínas Virales , Animales , Peste Porcina Clásica/inmunología , Peste Porcina Clásica/metabolismo , Peste Porcina Clásica/virología , Virus de la Fiebre Porcina Clásica/enzimología , Virus de la Fiebre Porcina Clásica/inmunología , Virus de la Fiebre Porcina Clásica/metabolismo , Virus de la Fiebre Porcina Clásica/patogenicidad , Endopeptidasas/química , Endopeptidasas/metabolismo , Histona Desacetilasa 1/biosíntesis , Histona Desacetilasa 1/metabolismo , Factor 3 Regulador del Interferón , Proteínas de la Nucleocápside/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Factor de Transcripción Sp1/metabolismo , Porcinos/virología , Proteínas del Núcleo Viral/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Ubiquitinas/metabolismo , Citocinas/metabolismo , Virus de la Diarrea Epidémica Porcina/inmunología , Virus de la Diarrea Epidémica Porcina/metabolismo , Dominios ProteicosRESUMEN
Classical swine fever virus (CSFV), the etiological agent of classical swine fever (CSF), causes serious financial losses to the pig industry. Using yeast two-hybrid screening, we have previously identified ribosomal protein RPLP1 as a potential binding partner of CSFV NS4B. In this study, the interaction between host RPLP1 and CSFV NS4B was further characterized by co-immunoprecipitation (co-IP), glutathione S-transferase (GST) pulldown, and confocal microscopy. In addition, lentivirus-mediated shRNA knockdown of RPLP1 drastically attenuated CSFV growth, while stable overexpression of RPLP1 markedly enhanced CSFV production. Moreover, cellular RPLP1 expression was found to be significantly up-regulated along with CSFV infection. Dual-luciferase reporter assay showed that depletion of RPLP1 had no effects on the activity of CSFV internal ribosome entry site (IRES). In the first life cycle of CSFV, further studies revealed that RPLP1 depletion did not influence the intracellular viral RNA abundance but diminished the intracellular and extracellular progeny virus titers as well as the viral E2 protein expression, which indicates that RPLP1 is crucial for CSFV genome translation. In summary, this study demonstrated that RPLP1 interacts with CSFV NS4B and enhances virus production via promoting translation of viral genome.
Asunto(s)
Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica , Animales , Peste Porcina Clásica/metabolismo , Virus de la Fiebre Porcina Clásica/genética , Genoma Viral , Unión Proteica , Porcinos , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Replicación ViralRESUMEN
Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a highly contagious disease of swine with high morbidity and mortality that negatively affects the pig industry worldwide, in particular in China. Soon after the endocytosis of CSFV, the virus makes full use of the components of host cells to complete its life cycle. The endocytosis sorting complex required for transport (ESCRT) system is a central molecular machine for membrane protein sorting and scission in eukaryotic cells that plays an essential role in many physiological metabolic processes, including invasion and egress of envelope viruses. However, the molecular mechanism that ESCRT uses to regulate the replication of CSFV is unknown. In this study, we demonstrated that the ESCRT-I complex Tsg101 protein participates in clathrin-mediated endocytosis of CSFV and is also involved in CSFV trafficking. Tsg101 assists the virus in entering the host cell through the late endosome (Rab7 and Rab9) and finally reaching the lysosome (Lamp-1). Interestingly, Tsg101 is also involved in the viral replication process by interacting with nonstructural proteins 4B and 5B of CSFV. Finally, confocal microscopy showed that the replication complex of Tsg101 and double-stranded RNA (dsRNA) or NS4B and NS5B protein was close to the endoplasmic reticulum (ER), not the Golgi, in the cytoplasm. Collectively, our finding highlights that Tsg101 regulates the process of CSFV entry and replication, indicating that the ESCRT plays an important role in the life cycle of CSFV. Thus, ESCRT molecules could serve as therapeutic targets to combat CSFV infection.IMPORTANCE CSF, caused by CSFV, is a World Organization for Animal Health (OIE) notifiable disease and causes significant financial losses to the pig industry globally. The ESCRT machinery plays an important regulatory role in several members of the genera Flavivirus and Hepacivirus within the family Flaviviridae, such as hepatitis C virus, Japanese encephalitis virus, and dengue virus. Previous reports have shown that assembling and budding of these viruses require ESCRT. However, the role of ESCRT in Pestivirus infection remains to be elucidated. We determined the molecular mechanisms of the regulation of CSFV infection by the major subunit Tsg101 of ESCRT-I. Interestingly, Tsg101 plays an essential regulatory role in both clathrin-mediated endocytosis and genome replication of CSFV. Overall, the results of this study provide further insights into the molecular function of ESCRT-I complex protein Tsg101 during CSFV infection, which may serve as a molecular target for pestivirus inhibitors.
Asunto(s)
Virus de la Fiebre Porcina Clásica/fisiología , Proteínas de Unión al ADN/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Factores de Transcripción/metabolismo , Internalización del Virus , Replicación Viral , Animales , Línea Celular , Peste Porcina Clásica/metabolismo , Peste Porcina Clásica/virología , Proteínas de Unión al ADN/genética , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/virología , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Endosomas/metabolismo , Endosomas/virología , Interacciones Huésped-Patógeno , Lisosomas/metabolismo , Lisosomas/virología , ARN Viral/metabolismo , Porcinos , Factores de Transcripción/genética , Proteínas no Estructurales Virales/metabolismo , Compartimentos de Replicación Viral/metabolismoRESUMEN
Interactions between the major structural glycoprotein E2 of classical swine fever virus (CSFV) with host proteins have been identified as important factors affecting virus replication and virulence. Previously, using the yeast two-hybrid system, we identified swine host proteins specifically interacting with CSFV E2. In this report, we use a proximity ligation assay to demonstrate that swine host protein CCDC115 interacts with E2 in CSFV-infected swine cells. Using a randomly mutated E2 library in the context of a yeast two-hybrid methodology, specific amino acid mutations in the CSFV E2 protein responsible for disrupting the interaction with CCDC115 were identified. A recombinant CSFV mutant (E2ΔCCDC115v) harboring amino acid changes disrupting the E2 protein interaction with CCDC115 was produced and used as a tool to assess the role of the E2-CCDC115 interaction in viral replication and virulence in swine. CSFV E2ΔCCDC115v showed a slightly decreased ability to replicate in the SK6 swine cell line and a greater replication defect in primary swine macrophage cultures. A decreased E2-CCDC115 interaction detected by PLA is observed in cells infected with E2ΔCCDC115v. Importantly, animals intranasally infected with 105 TCID50 of E2ΔCCDC115v experienced a significantly longer survival period when compared with those infected with the parental Brescia strain. This result would indicate that the ability of CSFV E2 to bind host CCDC115 protein during infection plays an important role in virus replication in swine macrophages and in virus virulence during the infection in domestic swine.
Asunto(s)
Virus de la Fiebre Porcina Clásica/fisiología , Peste Porcina Clásica/virología , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Replicación Viral , Animales , Células Cultivadas , Peste Porcina Clásica/metabolismo , Peste Porcina Clásica/patología , Virus de la Fiebre Porcina Clásica/genética , Virus de la Fiebre Porcina Clásica/metabolismo , Virus de la Fiebre Porcina Clásica/patogenicidad , Interacciones Huésped-Patógeno , Macrófagos/virología , Mutación , Unión Proteica , Análisis de Supervivencia , Porcinos , Proteínas del Envoltorio Viral/genética , Virulencia/genética , Replicación Viral/genéticaRESUMEN
E2 is the major structural glycoprotein of the classical swine fever virus (CSFV). E2 has been shown to be involved in important virus functions such as replication and virulence in swine. Using the yeast two-hybrid system, we previously identified several host proteins specifically interacting with CSFV E2. Here, we analyze the protein interaction of E2 with SERTA domain containing protein 1 (SERTAD1), a factor involved in the stimulation of the transcriptional activities of different host genes. We have confirmed that the interaction between these two proteins occurs in CSFV-infected swine cells by using a proximity ligation assay and confocal microscopy. Amino acid residues in the CSFV E2 protein that are responsible for mediating the interaction with SERTAD1 were mapped by a yeast two-hybrid approach using a randomly mutated E2 library. Using that information, a recombinant CSFV mutant (E2ΔSERTAD1v) that harbors substitutions in those residues mediating the protein-interaction with SERTAD1 was developed and used to study the role of the E2-SERTAD1 interaction in viral replication and virulence in swine. CSFV E2ΔSERTAD1v, when compared to the parental BICv, showed a clearly decreased ability to replicate in the SK6 swine cell line and a more severe replication defect in primary swine macrophage cultures. Importantly, 80% of animals infected with E2ΔSERTAD1v survived infection, remaining clinically normal during the 21-day observational period. This result would indicate that the ability of CSFV E2 to bind host SERTAD1 protein during infection plays a critical role in virus virulence.
Asunto(s)
Virus de la Fiebre Porcina Clásica/fisiología , Peste Porcina Clásica/metabolismo , Peste Porcina Clásica/virología , Interacciones Huésped-Patógeno , Factores de Transcripción/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Replicación Viral , Animales , Línea Celular , Mutación , Unión Proteica , Porcinos , Técnicas del Sistema de Dos Híbridos , VirulenciaRESUMEN
Classical swine fever virus (CSFV) is one of the most important viral pathogens leading worldwide threats to pig industry. MicroRNAs (miRNAs) play important roles in regulating virus replication, but whether miRNAs affect CSFV infection is still poorly understood. In previous study, we identified four miRNAs that were down-regulated by CSFV in swine umbilical vein endothelial cells (SUVEC). In this study, miR-140, one of the most potently down-regulated genes was investigated. We found that the miRNA expression was significantly inhibited by CSFV infection. Subsequent studies revealed that miR-140 mimics signiï¬cantly inhibited CSFV replication, while the inhibition of endogenous miR-140 enhanced CSFV replication. By using bioinformatics prediction, luciferase reporter system, real-time fluorescence quantitative PCR (RT-qPCR) and Western blot assays, we further demonstrated that miR-140 bind to the 3' UTR of Rab25 mRNA to regulate its expression. We also analyzed the expression pattern of Rab25 in SUVECs after CSFV infection. The results showed that CSFV infection induced Rab25 expression. Finally, Rab25 was found to promote CSFV replication. In conclusion, this study demonstrated that CSFV inhibits miR-140 expression and miR-140 inhibits replication by binding to host factor Rab25.
Asunto(s)
Virus de la Fiebre Porcina Clásica/efectos de los fármacos , Células Endoteliales/metabolismo , MicroARNs/antagonistas & inhibidores , MicroARNs/metabolismo , Venas Umbilicales/metabolismo , Replicación Viral/efectos de los fármacos , Proteínas de Unión al GTP rab/metabolismo , Animales , Peste Porcina Clásica/metabolismo , Peste Porcina Clásica/virología , Virus de la Fiebre Porcina Clásica/patogenicidad , Regulación hacia Abajo , Células HEK293 , Humanos , Unión Proteica , ARN Mensajero/metabolismo , PorcinosRESUMEN
A wide range of endemic and epidemic viruses, including classic swine fever virus (CSFV), pseudorabies virus (PRV) and others, are among the most economically important pathogens in pigs and have severely affected the national economy, human health and animal welfare and productivity. The RSAD2 exhibits antiviral activity against various DNA and RNA viruses. In this study, we successfully accomplished site-specific insertion of the porcine RSAD2 gene (pRSAD2) at the porcine ROSA26 (pROSA26) locus, generating pRSAD2 gene knock-in (pRSAD2-KI) PK-15 cells and porcine foetal fibroblasts (PFFs) via CRISPR/Cas9 technology. Gene expression analysis confirmed that pRSAD2-KI cells stably and efficiently overexpressed the pRSAD2 gene. Furthermore, viral challenge studies in vitro indicated that site-specific integration of the pRSAD2 gene not only effectively reduced CSFV infection but also PRV infection. More importantly, we ultimately successfully produced a pRSAD2-KI pig that constitutively overexpressed the pRSAD2, viral challenge results indicated that fibroblasts isolated from the pRSAD2-KI pig reduced CSFV infection. Taken together, these results suggest that CRISPR/Cas9-mediated knock-in strategy can be used for producing pRSAD2-KI pigs.
Asunto(s)
Sistemas CRISPR-Cas , Técnicas de Sustitución del Gen , Proteínas Hierro-Azufre/genética , Transgenes , Animales , Proteína 9 Asociada a CRISPR , Línea Celular , Peste Porcina Clásica/metabolismo , Fibroblastos/virología , Expresión Génica , Genes Reporteros , Herpesvirus Suido 1/metabolismo , Porcinos , Replicación Viral/genéticaRESUMEN
In the host, many RING domain E3 ligases have been reported to inhibit viral replication through various mechanisms. In a previous screen, we found that porcine RING finger protein 114 (pRNF114), a RING domain E3 ubiquitin ligase, inhibits classical swine fever virus (CSFV) replication. This study aimed to clarify the underlying antiviral mechanism of pRNF114 against CSFV. Upon CSFV infection, pRNF114 mRNA was upregulated both in vitro and in vivo CSFV replication was significantly suppressed in PK-pRNF114 cells stably expressing pRNF114 by the lentivirus-delivered system, whereas CSFV growth was enhanced in PK-15 cells with RNF114 knockout by the CRISPR/Cas9 system. The RING domain of pRNF114, which has E3 ubiquitin ligase activity, is crucial for its antiviral activity. Mechanistically, pRNF114 interacted with the CSFV NS4B protein through their C-terminal domains, which led to the K27-linked polyubiquitination and degradation of NS4B through a proteasome-dependent pathway. Collectively, these findings indicate that pRNF114 as a critical regulator of CSFV replication and uncover a mechanism by which pRNF114 employs its E3 ubiquitin ligase activity to inhibit CSFV replication.IMPORTANCE Porcine RING finger protein 114 (pRNF114) is a member of the RING domain E3 ligases. In this study, it was shown that pRNF114 is a potential anti-CSFV factor and the anti-CSFV effect of pRNF114 depends on its E3 ligase activity. Notably, pRNF114 targets and catalyzes the K27-linked polyubiquitination of the NS4B protein and then promotes proteasome-dependent degradation of NS4B, inhibiting the replication of CSFV. To our knowledge, pRNF114 is the first E3 ligase to be identified as being involved in anti-CSFV activity, and targeting NS4B could be a crucial route for antiviral development.
Asunto(s)
Virus de la Fiebre Porcina Clásica/fisiología , Peste Porcina Clásica/prevención & control , Lisina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Proteínas no Estructurales Virales/metabolismo , Replicación Viral , Animales , Peste Porcina Clásica/metabolismo , Peste Porcina Clásica/virología , Células HEK293 , Humanos , Lisina/genética , Porcinos , Ubiquitina-Proteína Ligasas/genética , Proteínas no Estructurales Virales/genéticaRESUMEN
Genetic labelling of viruses with a fluorophore allows to study their life cycle in real time, without the need for fixation or staining techniques. Within the family Flaviviridae, options for genetic labelling of non-structural proteins exist. Yet, no system to genetically label structural proteins has been put forward to date. Taking advantage of a previously described site within the structural protein E2, a fluorophore was introduced into a cytopathogenic (cpe) BVDV-1 virus (BVDVE2_fluo). This insertion was well tolerated, resulting in a 2-fold drop in titer compared to the parental virus, and remained stably integrated into the genome for more than 10 passages. The fluorophore E2 fusion protein was readily detectable in purified virus particles by Western blot and fluorescence microscopy and the particle integrity and morphology was confirmed by cryo electron microscopy. The same integration site could also be used to label the related Classical swine fever virus. Also, BVDVE2_fluo particles bound to fluorophore labelled CD46 expressing cells could be resolved in fluorescence microscopy. This underlines the applicability of BVDVE2_fluo as a tool to study the dynamics of the whole life cycle of BVDV in real time.
Asunto(s)
Diarrea Mucosa Bovina Viral/virología , Virus de la Diarrea Viral Bovina , Microscopía Fluorescente , Proteínas del Envoltorio Viral , Animales , Diarrea Mucosa Bovina Viral/metabolismo , Diarrea Mucosa Bovina Viral/patología , Bovinos , Línea Celular , Peste Porcina Clásica/metabolismo , Peste Porcina Clásica/patología , Peste Porcina Clásica/virología , Virus de la Fiebre Porcina Clásica/genética , Virus de la Fiebre Porcina Clásica/metabolismo , Microscopía por Crioelectrón , Virus de la Diarrea Viral Bovina/genética , Virus de la Diarrea Viral Bovina/metabolismo , Proteína Cofactora de Membrana/metabolismo , Microscopía Fluorescente/métodos , Porcinos , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Virión/metabolismoRESUMEN
Rab1A belongs to the small Rab GTPase family and is involved in the lifecycle of numerous viruses. Here, knockdown of Rab1A inhibited CSFV growth. Further study revealed that Rab1A depletion decreased intracellular and extracellular CSFV titers, but did not affect intracellular virus genome copies and E2 protein expression within a virus lifecycle, which suggested that Rab1A is required for CSFV particle assembly rather than for genome replication or virion release. This was proofed by blocking the spread of virus using neutralizing antibodies, through which the negative effects of Rab1A knockdown on multi-cycle replication of CSFV were eliminated. Moreover, co-immunoprecipitation and confocal microscopy assays showed that Rab1A bound to CSFV NS5A protein, indicating that Rab1A and viral NS5A proteins may work cooperatively during CSFV particle assembly. In conclusion, this study demonstrated for the first time that Rab1A is required for CSFV particle assembly and binds to viral particle assembly-related NS5A protein.
Asunto(s)
Virus de la Fiebre Porcina Clásica/fisiología , Peste Porcina Clásica/metabolismo , Ensamble de Virus , Proteínas de Unión al GTP rab1/metabolismo , Animales , Peste Porcina Clásica/genética , Peste Porcina Clásica/virología , Virus de la Fiebre Porcina Clásica/genética , Interacciones Huésped-Patógeno , Porcinos , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Replicación Viral , Proteínas de Unión al GTP rab1/genéticaRESUMEN
BACKGROUND: Classical swine fever virus (CSFV) is the causative pathogen of Classical swine fever (CSF), a highly contagious disease of swine. Viperin is one of the hundreds of interferon-stimulated genes (ISGs), and possesses a wide range of antiviral activities. The aim of this study was to explore whether porcine Viperin has the anti-CSFV activity. METHOD: The influences of CSFV infection on Viperin expression and Newcastle disease virus (NDV)/Pseudorabies virus (PRV)-induced Viperin expression were examined in 3D4/21 cells and porcine peripheral blood mononuclear cells (PBMCs). Porcine Viperin gene was amplified to generate cell line PK-Vi over-expressing Viperin. CSFV was inoculated in the cell lines and viral load was detected by qRT-PCR, virus titration and Western blot. The influence of Viperin expression on CSFV binding, entry and release in the cells was also examined. The co-localization of Viperin with CSFV and its proteins (E2, NS5B) was determined by confocal laser scanning microscopy test. Co-IP assay was performed to check the interaction of Viperin with CSFV proteins. RESULTS: CSFV infection could not induce Viperin expression in vitro while significantly inhibiting NDV/PRV-induced Viperin expression at 12, 24 and 48 h post infection (hpi; P < 0.05). The proliferation of CSFV in PK-Vi was significantly inhibited at 24, 48 and 72 hpi (P < 0.05), comparing with control cells (PK-C1 expressing EGFP). Virus in both cell culture supernatants and cell pellets were reduced equally. CSFV binding and entry in the cells were not interfered by Viperin expression. These results indicated its anti-CSFV function occurred during the genome and/or protein synthesis step. Confocal laser scanning microscopy test showed the Viperin-EGFP protein co-localized with CSFV E2 protein in CSFV infected PK-Vi cells. Further experiments indicated that Viperin protein co-localized with E2 and NS5B proteins of CSFV in the transfected 293 T cells. Furthermore, Co-IP assay confirmed the interaction of Viperin with E2 protein, but not NS5B. CONCLUSION: Porcine Viperin effectively inhibited CSFV replication in vitro, potentially via the interaction of Viperin with CSFV E2 protein in cytoplasm. The results provided foundation for further studies of the interaction of Viperin with CSFV and other viruses.
Asunto(s)
Virus de la Fiebre Porcina Clásica/fisiología , Proteínas/metabolismo , Replicación Viral , Animales , Células Cultivadas , Peste Porcina Clásica/metabolismo , Peste Porcina Clásica/virología , Virus de la Fiebre Porcina Clásica/efectos de los fármacos , Expresión Génica , Humanos , Leucocitos Mononucleares/virología , Plásmidos/genética , Unión Proteica , Proteínas/genética , Proteínas/farmacología , Porcinos , Proteínas Virales/metabolismo , Acoplamiento Viral , Internalización del Virus , Liberación del Virus , Replicación Viral/efectos de los fármacosRESUMEN
Classical swine fever (CSF) is a severe, febrile and highly contagious disease caused by classical swine fever virus (CSFV) that has resulted in huge economic losses in the pig industry worldwide. CSFV Npro has been actively studied but remains incompletely understood. Few studies have investigated the cellular proteins that interact with Npro and their participation in viral replication. Here, the yeast two-hybrid (Y2H) system was employed to screen Npro-interacting proteins from a porcine alveolar macrophage (PAM) cDNA library, and a blast search of the NCBI database revealed that 15 cellular proteins interact with Npro. The interaction of Npro with ribosomal protein S20, also known as universal S10 (uS10), was further confirmed by co-immunoprecipitation and glutathione S-transferase pull-down assays. Furthermore, uS10 overexpression inhibited CSFV replication, whereas the knockdown of uS10 promoted CSFV replication in PAMs. In addition, Npro or CSFV reduced uS10 expression in PAMs in a proteasome-dependent manner, indicating that Npro-uS10 interaction might contribute to persistent CSFV replication. Our previous research showed that CSFV decreases Toll-like receptor 3 (TLR3) expression. The results showed that uS10 knockdown reduced TLR3 expression, and that uS10 overexpression increased TLR3 expression. Notably, uS10 knockdown did not promote CSFV replication following TLR3 overexpression. Conversely, uS10 overexpression did not inhibit CSFV replication following TLR3 knockdown. These results revealed that uS10 inhibits CSFV replication by modulating TLR3 expression. This work addresses a novel aspect of the regulation of the innate antiviral immune response during CSFV infection.
Asunto(s)
Virus de la Fiebre Porcina Clásica/fisiología , Peste Porcina Clásica/metabolismo , Endopeptidasas/metabolismo , Proteínas Ribosómicas/metabolismo , Proteínas Virales/metabolismo , Replicación Viral , Animales , Peste Porcina Clásica/genética , Peste Porcina Clásica/virología , Virus de la Fiebre Porcina Clásica/genética , Endopeptidasas/genética , Unión Proteica , Proteínas Ribosómicas/genética , Porcinos , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/metabolismo , Proteínas Virales/genéticaRESUMEN
Classical swine fever virus (CSFV), which causes typical clinical characteristics in piglets, including hemorrhagic syndrome and immunosuppression, is linked to hepatitis C and dengue virus. Oxidative stress and a reduced mitochondrial transmembrane potential are disturbed in CSFV-infected cells. The balance of mitochondrial dynamics is essential for cellular homeostasis. In this study, we offer the first evidence that CSFV induces mitochondrial fission and mitophagy to inhibit host cell apoptosis for persistent infection. The formation of mitophagosomes and decline in mitochondrial mass relevant to mitophagy were detected in CSFV-infected cells. CSFV infection increased the expression and mitochondrial translocation of Pink and Parkin. Upon activation of the PINK1 and Parkin pathways, Mitofusin 2 (MFN2), a mitochondrial fusion mediator, was ubiquitinated and degraded in CSFV-infected cells. Mitophagosomes and mitophagolysosomes induced by CSFV were, respectively, observed by the colocalization of LC3-associated mitochondria with Parkin or lysosomes. In addition, a sensitive dual fluorescence reporter (mito-mRFP-EGFP) was utilized to analyze the delivery of mitophagosomes to lysosomes. Mitochondrial fission caused by CSFV infection was further determined by mitochondrial fragmentation and Drp1 translocation into mitochondria using a confocal microscope. The preservation of mitochondrial proteins, upregulated apoptotic signals and decline of viral replication resulting from the silencing of Drp1 and Parkin in CSFV-infected cells suggested that CSFV induced mitochondrial fission and mitophagy to enhance cell survival and viral persistence. Our data for mitochondrial fission and selective mitophagy in CSFV-infected cells reveal a unique view of the pathogenesis of CSFV infection and provide new avenues for the development of antiviral strategies.
Asunto(s)
Apoptosis , Virus de la Fiebre Porcina Clásica/fisiología , Peste Porcina Clásica/metabolismo , Peste Porcina Clásica/virología , Mitocondrias/metabolismo , Dinámicas Mitocondriales , Mitofagia , Animales , Línea Celular , Supervivencia Celular , Mitocondrias/ultraestructura , Proteínas Mitocondriales/metabolismo , Fagosomas/metabolismo , Porcinos , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Replicación ViralRESUMEN
Viral coinfection or superinfection in host has caused public health concern and huge economic losses of farming industry. The influence of viral coinfection on cellular protein abundance is essential for viral pathogenesis. Based on a coinfection model for porcine circovirus type 2 (PCV2) and classical swine fever virus (CSFV) developed previously by our laboratory, isobaric tags for relative and absolute quantitation (iTRAQ)-coupled LC-MS/MS proteomic profiling was performed to explore the host cell responses to PCV2-CSFV coinfection. Totally, 3932 proteins were identified in three independent mass spectrometry analyses. Compared with uninfected cells, 304 proteins increased (fold change >1.2) and 198 decreased (fold change <0.833) their abundance in PCV2-infected cells (p < 0.05), 60 and 61 were more and less abundant in CSFV-infected cells, and 196 and 158 were more and less abundant, respectively in cells coinfected with PCV2 and CSFV. Representative differentially abundant proteins were validated by quantitative real-time PCR, Western blotting and confocal laser scanning microscopy. Bioinformatic analyses confirmed the dominant role of PCV2, and indicated that mitochondrial dysfunction, nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated oxidative stress response and apoptosis signaling pathways might be the specifical targets during PCV2-CSFV coinfection.
Asunto(s)
Cromatografía Liquida/métodos , Infecciones por Circoviridae/metabolismo , Circovirus/química , Virus de la Fiebre Porcina Clásica/química , Peste Porcina Clásica/metabolismo , Coinfección/metabolismo , Proteínas Virales/análisis , Animales , Línea Celular , Circovirus/patogenicidad , Virus de la Fiebre Porcina Clásica/patogenicidad , Análisis por Conglomerados , Marcaje Isotópico , Modelos Biológicos , Porcinos , Espectrometría de Masas en Tándem/métodos , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
BACKGROUND: Classical swine fever (CSF) caused by CSF virus (CSFV) is a highly contagious disease of the pigs. A number of studies have suggested that CSFV non-structural (NS) 5A protein is involved in CSFV-associated pathogenesis, but its mechanism is still uncertain. The aim of this study was to investigate the roles of NS5A protein in CSFV-associated pathogenesis in cultured porcine alveolar macrophages (PAMs). METHODS: After PAMs cultured in vitro were transfected with CSFV NS5A, the alterations in IL-1ß, IL-6 and TNF-α expression were determined by ELISA, the RIG-I signaling activity related to inflammatory cytokine secretion was investigated by Western blot and Immunofluorescent staining. RESULTS: It was suggested that, the stable expressed CSFV NS5A solely had no influence on the expressions of inflammatory cytokines IL-1ß, IL-6 and TNF-α in PAMs Moreover, NS5A protein could suppressed IL-1ß, IL-6 and TNF-α expression induced by poly(I:C). It was also showed that NS5A protein did not impair the expressions of RIG-I, MDA5, IPS-1, NF-κB and IkBα in cells without poly(I:C) stimulation. Protein expressions of RIG-I, MDA5, IPS-1, NF-κB were not disrupted by NS5A protein in poly(I:C)-stimulated cells, while poly(I:C)-induced NF-κB nuclear translocation and activity was obviously suppressed by this protein. A suppression in poly(I:C)-induced IkBα degradation in NS5A-expressing cells was also observed. CONCLUSION: These data indicated that CSFV NS5A protein could inhibit the secretion of inflammatory cytokine induced by poly(I:C) through the suppression of the NF-κB signaling pathway, indicating the participation of CSFV NS5A protein in the pathogenesis of CSFV.
Asunto(s)
Virus de la Fiebre Porcina Clásica/metabolismo , Peste Porcina Clásica/virología , Citocinas/metabolismo , Macrófagos Alveolares/metabolismo , FN-kappa B/metabolismo , Proteínas no Estructurales Virales/metabolismo , Animales , Peste Porcina Clásica/genética , Peste Porcina Clásica/metabolismo , Virus de la Fiebre Porcina Clásica/genética , Citocinas/genética , Interacciones Huésped-Parásitos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , FN-kappa B/genética , Transducción de Señal , Porcinos , Proteínas no Estructurales Virales/genéticaRESUMEN
UNLABELLED: Many viruses trigger the type I interferon (IFN) pathway upon infection, resulting in the transcription of hundreds of interferon-stimulated genes (ISGs), which define the antiviral state of the host. Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious viral disease endangering the pig industry in many countries. However, anti-CSFV ISGs are poorly documented. Here we screened 20 ISGs that are commonly induced by type I IFNs against CSFV in lentivirus-delivered cell lines, resulting in the identification of guanylate-binding protein 1 (GBP1) as a potent anti-CSFV ISG. We observed that overexpression of GBP1, an IFN-induced GTPase, remarkably suppressed CSFV replication, whereas knockdown of endogenous GBP1 expression by small interfering RNAs significantly promoted CSFV growth. Furthermore, we demonstrated that GBP1 acted mainly on the early phase of CSFV replication and inhibited the translation efficiency of the internal ribosome entry site of CSFV. In addition, we found that GBP1 was upregulated at the transcriptional level in CSFV-infected PK-15 cells and in various organs of CSFV-infected pigs. Coimmunoprecipitation and glutathione S-transferase (GST) pulldown assays revealed that GBP1 interacted with the NS5A protein of CSFV, and this interaction was mapped in the N-terminal globular GTPase domain of GBP1. Interestingly, the K51 of GBP1, which is crucial for its GTPase activity, was essential for the inhibition of CSFV replication. We showed further that the NS5A-GBP1 interaction inhibited GTPase activity, which was critical for its antiviral effect. Taking our findings together, GBP1 is an anti-CSFV ISG whose action depends on its GTPase activity. IMPORTANCE: Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), an economically important viral disease affecting the pig industry in many countries. To date, only a few host restriction factors against CSFV, including interferon-stimulated genes (ISGs), have been characterized. Using a minilibrary of porcine ISGs, we identify porcine guanylate-binding protein 1 (GBP1) as a potent antiviral ISG against CSFV. We further show that the anti-CSFV action of GBP1 depends on its GTPase activity. The K51 of GBP1, critical for its GTPase activity, is essential for the antiviral action of GBP1 against CSFV replication, and the binding of the NS5A protein to GBP1 antagonizes the GTPase activity and thus the antiviral effect. This study will facilitate the development of anti-CSFV therapeutic agents by targeting host factors and may provide a new strategy for the control of CSF.